Quality Control
Sample clustering
PCA
## PC1 PC2 group condition replicate name
## WT_4 -32.90151 -5.235742 Control:1 Control 1 WT_4
## WT_5 -28.67289 -8.954453 Control:2 Control 2 WT_5
## WT_6 -41.17100 10.740516 Control:3 Control 3 WT_6
## S926glc4 39.03169 -1.852210 Mutant_LOF:1 Mutant_LOF 1 S926glc4
## S926glc5 24.52983 8.343747 Mutant_LOF:2 Mutant_LOF 2 S926glc5
## S926glc6 39.18388 -3.041858 Mutant_LOF:3 Mutant_LOF 3 S926glc6
Number of genes detected
rRNA percentage
Proportions of gene biotypes
Remove rRNA from the analysis
dds = dds[!(row.names(counts(dds)) %in% ensembl.genes$gene_id[ensembl.genes$gene_biotype %in% c("rRNA", "snoRNA", "snRNA")]),]
dds = dds[rowSums(counts(dds)) > 0,]Check rRNA removed
Check chrM intact
##
## chr2L chr2R chr3L chr3R chr4 chrX chrY chrM
## 2902 3172 2956 3594 91 2326 42 38Clustering after rRNA removal
PCA after rRNA removal
## PC1 PC2 group condition replicate name
## WT_4 -32.87163 -5.240295 Control:1 Control 1 WT_4
## WT_5 -28.64340 -8.957790 Control:2 Control 2 WT_5
## WT_6 -41.13673 10.755695 Control:3 Control 3 WT_6
## S926glc4 38.99282 -1.833142 Mutant_LOF:1 Mutant_LOF 1 S926glc4
## S926glc5 24.51421 8.298705 Mutant_LOF:2 Mutant_LOF 2 S926glc5
## S926glc6 39.14474 -3.023172 Mutant_LOF:3 Mutant_LOF 3 S926glc6
Size Factors
## WT_4 WT_5 WT_6 S926glc4 S926glc5 S926glc6
## 1.0398668 1.0648837 1.1695855 0.8627809 0.9688625 0.9497324
MA Plots
## [1] 1
## [1] 2
## [1] 3
## [1] 2
## [1] 3
## [1] 1
## [1] 2
## [1] 3
## [1] 2
## [1] 3
Normalised counts (boxplot)
| seqnames | start | end | width | strand | gene_id | gene_biotype | entrezgene_id | external_gene_name | |
|---|---|---|---|---|---|---|---|---|---|
| FBgn0000559 | chr2L | 21677913 | 21682164 | 4252 |
|
FBgn0000559 | protein_coding | 35422 | eEF2 |
| FBgn0013674 | chrM | 1474 | 3009 | 1536 |
|
FBgn0013674 | protein_coding | 192469 | mt:CoI |
| FBgn0013676 | chrM | 4736 | 5524 | 789 |
|
FBgn0013676 | protein_coding | 192471 | mt:CoIII |
| FBgn0013678 | chrM | 10499 | 11635 | 1137 |
|
FBgn0013678 | protein_coding | 192472 | mt:Cyt-b |
| FBgn0284245 | chr2R | 11891135 | 11895762 | 4628 |
|
FBgn0284245 | protein_coding | 36271 | eEF1alpha1 |
These all seem to be protein-coding genes, so we will not remove them.
Normalised counts (Transcripts per million (TPM))
| seqnames | start | end | width | strand | gene_id | gene_biotype | entrezgene_id | external_gene_name | |
|---|---|---|---|---|---|---|---|---|---|
| FBgn0066084 | chr2R | 24903416 | 24903935 | 520 |
|
FBgn0066084 | protein_coding | 251466 | RpL41 |
These all seem to be protein-coding genes, so we will not remove them.
Session Info
## R version 4.0.5 (2021-03-31)
## Platform: x86_64-apple-darwin17.0 (64-bit)
## Running under: macOS Big Sur 10.16
##
## Matrix products: default
## BLAS: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRblas.dylib
## LAPACK: /Library/Frameworks/R.framework/Versions/4.0/Resources/lib/libRlapack.dylib
##
## locale:
## [1] en_US.UTF-8/en_US.UTF-8/en_US.UTF-8/C/en_US.UTF-8/en_US.UTF-8
##
## attached base packages:
## [1] parallel stats4 stats graphics grDevices utils datasets
## [8] methods base
##
## other attached packages:
## [1] kableExtra_1.3.4 Biostrings_2.58.0
## [3] XVector_0.30.0 scales_1.1.1
## [5] reshape2_1.4.4 knitr_1.31
## [7] biomaRt_2.46.3 GenomicFeatures_1.42.3
## [9] AnnotationDbi_1.52.0 genefilter_1.72.1
## [11] ggplot2_3.3.3 DESeq2_1.30.1
## [13] SummarizedExperiment_1.20.0 Biobase_2.50.0
## [15] MatrixGenerics_1.2.1 matrixStats_0.58.0
## [17] GenomicRanges_1.42.0 GenomeInfoDb_1.26.7
## [19] IRanges_2.24.1 S4Vectors_0.28.1
## [21] BiocGenerics_0.36.0
##
## loaded via a namespace (and not attached):
## [1] nlme_3.1-152 bitops_1.0-6 bit64_4.0.5
## [4] webshot_0.5.2 RColorBrewer_1.1-2 progress_1.2.2
## [7] httr_1.4.2 rprojroot_2.0.2 tools_4.0.5
## [10] bslib_0.2.4 utf8_1.2.1 R6_2.5.0
## [13] mgcv_1.8-34 DBI_1.1.1 colorspace_2.0-0
## [16] withr_2.4.1 tidyselect_1.1.0 prettyunits_1.1.1
## [19] bit_4.0.4 curl_4.3 compiler_4.0.5
## [22] rvest_1.0.0 xml2_1.3.2 DelayedArray_0.16.3
## [25] labeling_0.4.2 rtracklayer_1.50.0 sass_0.3.1
## [28] askpass_1.1 rappdirs_0.3.3 systemfonts_1.0.1
## [31] stringr_1.4.0 digest_0.6.27 Rsamtools_2.6.0
## [34] svglite_2.0.0 rmarkdown_2.7 pkgconfig_2.0.3
## [37] htmltools_0.5.1.1 highr_0.8 dbplyr_2.1.1
## [40] fastmap_1.1.0 rlang_0.4.10 rstudioapi_0.13
## [43] RSQLite_2.2.6 farver_2.1.0 jquerylib_0.1.3
## [46] generics_0.1.0 jsonlite_1.7.2 BiocParallel_1.24.1
## [49] dplyr_1.0.5 RCurl_1.98-1.3 magrittr_2.0.1
## [52] GenomeInfoDbData_1.2.4 Matrix_1.3-2 Rcpp_1.0.6
## [55] munsell_0.5.0 fansi_0.4.2 lifecycle_1.0.0
## [58] stringi_1.5.3 yaml_2.2.1 zlibbioc_1.36.0
## [61] plyr_1.8.6 BiocFileCache_1.14.0 grid_4.0.5
## [64] blob_1.2.1 crayon_1.4.1 lattice_0.20-41
## [67] splines_4.0.5 annotate_1.68.0 hms_1.0.0
## [70] locfit_1.5-9.4 pillar_1.6.0 codetools_0.2-18
## [73] geneplotter_1.68.0 XML_3.99-0.6 glue_1.4.2
## [76] evaluate_0.14 vctrs_0.3.7 gtable_0.3.0
## [79] openssl_1.4.3 purrr_0.3.4 assertthat_0.2.1
## [82] cachem_1.0.4 xfun_0.22 xtable_1.8-4
## [85] viridisLite_0.4.0 survival_3.2-10 tibble_3.1.0
## [88] GenomicAlignments_1.26.0 memoise_2.0.0 ellipsis_0.3.1